Dual Chemotherapeutic Loading in Oxalate Transferrin-Conjugated Polymersomes Incorporated into Chitosan Hydrogels for Site-Specific Targeting of Melanoma Cells.

In this work, we developed a smart drug delivery system composed of poly (ethylene glycol)-block-poly (ε-caprolactone) (PEG-PCL)-based polymersomes (Ps) loaded with doxorubicin (DOX) and vemurafenib (VEM). To enhance targeted delivery to malignant melanoma cells, these drug-loaded nanovesicles were conjugated to the oxalate transferrin variant (oxalate Tf) and incorporated into three-dimensional chitosan hydrogels. This innovative approach represents the first application of oxalate Tf for the precision delivery of drug-loaded polymersomes within a semi-solid dosage form based on chitosan hydrogels. These resulting semi-solids exhibited a sustained release profile for both encapsulated drugs. To evaluate their potency, we compared the cytotoxicity of native Tf-Ps with oxalate Tf-Ps. Notably, the oxalate Tf-Ps demonstrated a 3-fold decrease in cell viability against melanoma cells compared to normal cells and were 1.6-fold more potent than native Tf-Ps, indicating the greater potency of this nanoformulation. These findings suggest that dual-drug delivery using an oxalate-Tf-targeting ligand significantly enhances the drug delivery efficiency of Tf-conjugated nanovesicles and offers a promising strategy to overcome the challenge of multidrug resistance in melanoma therapy.

Targeted Therapy With Vemurafenib in Brazilian Children With Refractory Langerhans Cell Histiocytosis: Two Case Reports and Review of Literature.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a clonal myeloid neoplasm with inflammatory component. Refractory disease is a challenge, but vemurafenib has emerged as a therapeutic option. We will delineate the cases of two Brazilian children suffering from refractory LCH with a positive response to vemurafenib. CASES: Both cases had a diagnosis of multisystem disease with involvement of organs at risk and had not responded to standard and second-line treatment. After refractoriness to classic treatment regimens, the BRAF mutation was investigated and found to be positive in both patients, and target therapy with vemurafenib was sought. The first case has been using vemurafenib for about 2 years and the second case has been using it for about 3 years, having had an attempt to suspend the medication after concomitant use with maintenance therapy. However, the disease returned 4 months after stopping the medication. Fortunately, the disease returned to remission status after the medication was reintroduced. CONCLUSION: These cases represent the first reported instances of off-label vemurafenib use in Brazil for the treatment of LCH and both patients have demonstrated excellent responses to the medication. However, the long-term side effects are unknown in children, and prospective studies are needed. In addition, there is a lack of epidemiological data on histiocytosis in Brazil and studies evaluating the budgetary impact of incorporating BRAF mutation research and the use of vemurafenib into the public health system. These reports could be a starting point.

Estudo da encapsulação de fármacos antineoplásicos em sistemas nanoestruturados: Caracterização estrutural e efeito sinérgico de fármacos / Study of the encapsulation of antineoplastic drugs in nanostructured systems: Structured characterization and synergistic effect of drugs

Dentro da área da nanotecnologia, o sistema drug delivery vem sendo amplamente utilizado, cujo objetivo é proporcionar uma maior eficácia dos ativos farmacêuticos, podendo envolver desde uma distribuição mais seletiva dentro do organismo até a taxa que as moléculas serão liberadas e/ou a atenuação dos efeitos adversos provocados. Para isso, os ativos são encapsulados em nanoestruturas, podendo estas serem de natureza sintética ou natural. Dentre os nanocarreadores promissores encontram-se os cubossomos, que são nanoestruturas complexas capazes de encapsular ativos tanto hidrofílicos quanto hidrofóbicos. O objetivo deste projeto foi estudar a encapsulação de fármacos antineoplásicos em sistemas drug delivery contra linhagens celulares, investigando também as alterações estruturais sofridas pelos cubossomos e os efeitos sinérgicos dos fármacos, sendo eles: a doxorrubicina, a cisplatina, a vemurafenibe e a curcumina. As metodologias empregadas para elucidar o efeito das combinações dos fármacos, a estruturação da nanopartícula e sua citotoxicidade foram: os estudos de viabilidade celular pós-exposição, espalhamento dinâmico de luz, potencial zeta, análise de rastreamento de nanopartículas, espalhamento de raios-x a baixos ângulos, criomicroscopia eletrônica de transmissão, eficiência de encapsulação e ensaio de liberação. Inicialmente os fármacos foram testados isoladamente e em duplas, sendo utilizadas cinco linhagens celulares, afim de se promover um delineamento aos ensaios futuros. A partir destes resultados, foi-se optado por manter duas linhagens celulares, a HeLa, como representante de tecidos tumorais, e a HaCat, modelo de tecido saudável, devido a menor resistência apresentada por elas. Em relação as combinações entre as drogas, pode-se observar que todas as duplas formadas apresentaram resultados sinérgicos na linhagem tumoral, sendo mantida para os testes seguintes a combinação curcumina e vemurafenibe. Os cubossomos foram sintetizados eficientemente, sendo produzidos na ausência de fármacos bem como contendo curcumina e vemurafenibe. As nanopartículas apresentaram uma variação de diâmetro entre 189 ± 3 nm e 224 ± 2 nm, sendo o PDI entre 0,08 e 0,25. A conformação do cubossomo foi confirmada através da criomicroscopia eletrônica de transmissão e pelo espalhamento de raios-x a baixos ângulos, onde foi determinada uma estruturação característica de Pn3m. Para a eficiência de encapsulação os valores variaram entre 79% de encapsulação para a curcumina e 72% para a vemurafenibe, quando utilizadas isoladamente. No caso da encapsulação em dupla, os valores se converteram para 63% e 53% para a curcumina e vemurafenibe, respectivamente. A liberação das drogas do interior da nanopartícula oscilou entre 1500, 480 e 420 minutos para os cubossomos de curcumina, vemurafenibe e curcumina + vemunafenibe, respectivamente. Os testes de citotoxicidade demonstraram que as concentrações de 0,01 e 0,03 mg/mL foram capazes de promover uma viabilidade acima de 70%, porém, utilizando estas proporções não foi possível observar resultados significativos. Por fim, o sistema se mostrou estável e homogêneo, sendo capaz de promover a encapsulação dos fármacos tanto singularmente quanto em dupla e, apesar da quantidade de fármacos não ter sido suficiente para ocasionar alterações ao sistema celular, a execução deste trabalho abre portas para que novos estudos sejam realizados, podendo-se testar diferentes ativos bem como alterando a composição da nanopartícula afim de se reduzir a citotoxicidade

Within the area of nanotechnology, the drug delivery system has been widely used, whose objective is to provide greater effectiveness of pharmaceutical active ingredients, which may range from a more selective distribution within the organism to the rate at which the molecules will be released and/or the attenuation of adverse effects caused. To achieve this, the active ingredients are encapsulated in nanostructures, which may be synthetic or natural in nature. Among the promising nanocarriers are cubosomes, which are complex nanostructures capable of encapsulating both hydrophilic and hydrophobic active ingredients. The objective of this project was to study the encapsulation of antineoplastic drugs in drug delivery systems against cell lines, also investigating the structural changes undergone by the cubosomes and the synergistic effects ofthe drugs, namely: doxorubicin, cisplatin, vemurafenib and curcumin. The methodologies used to elucidate the effect of drug combinations, the structuring of the nanoparticle and its cytotoxicity were: post-exposure cell viability studies, dynamic light scattering, zeta potential, nanoparticle tracking analysis, small angle x-rays scattering, transmission electron cryomicroscopy, encapsulation efficiency and release assay. Initially, the drugs were tested alone and in pairs, using five cell lines, in order to promote a design for future trials. Based on these results, it was decided to maintain two cell lines, HeLa, as a representative oftumor tissues, and HaCat, a model ofhealthy tissue, due to their lower resistance. Regarding the combinations between the drugs, it can be observed that all the pairs formed presented synergistic results in the tumor lineage, with the combination of curcumin and vemurafenib being maintained for the following tests. Cubosomes were efficiently synthesized, being produced in the absence of drugs as well as containing curcumin and vemurafenib. The nanoparticles varied in diameter between 189 ± 3 nm and 224 ± 2 nm, with the PDI being between 0.08 and 0.25. The conformation ofthe cubosome was confirmed through transmission electron cryomicroscopy and small angle x-rays scattering, where a characteristic structure of Pn3m was determined. For encapsulation efficiency, values varied between 79% encapsulation for curcumin and 72% for vemurafenib, when used alone. ln the case of double encapsulation, the values converted to 63% and 53% for curcumin and vemurafenib, respectively. The release of drugs from the interior of the nanoparticle ranged between 1500, 480 and 420 minutes for the curcumin, vemurafenib and cubosomes with curcumin + vemunafenib, respectively. Cytotoxicity tests demonstrated that concentrations of 0.01 and 0.03 mg/mL were capable of promoting viability above 70%, however, using these proportions it was not possible to observe significant results. Finally, the system proved to be stable and homogeneous, being able to promote the encapsulation of drugs both singly and in pairs and, although the quantity of drugs was not enough to cause changes to the cellular system, the execution of this work opens doors for new studies are carried out, with the possibility oftesting different active ingredients as well as changing the composition of the nanoparticle in order to reduce cytotoxicity

Violacein improves vemurafenib response in melanoma spheroids.

Chemotherapy resistance is one of the main challenges in melanoma treatment. Violacein, a natural pigment produced by Chromobacterium violaceum, induces apoptosis in a variety of tumours, including melanoma. Here, we used BRAF-mutated melanoma spheroids to test the potential of violacein as a sensitizer of cellular viability and levels of the proteins p62 and fatty acid synthase (FASN). Importantly, violacein in combination with vemurafenib (ViVe) was able to interfere with spheroid survival at subtoxic concentrations. The results demonstrated that the ViVe protocol triggered cell death assessed by calcein and ethidium homodimer dyes. Accordingly, melanoma cells in 2D systems also showed a higher apoptosis rate when treated with ViVe. In the current study, we show evidence that ViVe downregulates crucial mediators like FASN, which partially explains how it acts as a sensitizer and ultimately improves the effectiveness of vemurafenib against melanoma cells.

Melanoma cells with acquired resistance to vemurafenib have decreased autophagic flux and display enhanced ability to transfer resistance.

Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAFV600E mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC50 (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells.

3-Bromopyruvate Suppresses the Malignant Phenotype of Vemurafenib-Resistant Melanoma Cells.

(1) BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.

Upregulation of the novel lncRNA U731166 is associated with migration, invasion and vemurafenib resistance in melanoma.

Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis-related processes. In this sense, we found the intergenic lncRNA U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA U73166 for cancer processes. Additionally, using naïve and vemurafenib-resistant cell lines and data from a patient before and after resistance, we found that vemurafenib-resistant samples had a higher expression of lncRNA U73166. Also, we retrieved data from the literature that indicates lncRNA U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA U73166 in metastasis development. We also pointed herein the lncRNA U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.

[Targeted therapies with BRAF inhibitors for pediatric low- and high-grade gliomas with BRAFv600e mutation. Prof. Dr. Juan P. Garrahan Hospital experience]. / Terapias dirigidas con inhibidores del BRAF en gliomas pediátricos de alto y bajo grado con la mutación BRAFV600E. Experiencia del Hospital Prof. Dr. Juan P. Garrahan.

The BRAFV600E point mutation plays a key role in the tumorigenesis of many gliomas. Inhibiting its product is part of the innovative therapies emerging in recent years. Knowing the role of these treatments is essential. The aim of this experience was to describe the clinical-radiological response of pediatric BRAFV600E mutated gliomas treated with BRAF inhibitors. To this end, a descriptive and retrospective study was performed in patients under 16 years of age with BRAFV600E gliomas, who received vemurafenib or dabrafenib at Hospital Garrahan. Thirteen patients treated in the last 7 years were included: 9 were low-grade and 4 high-grade gliomas. The median age at diagnosis was 8.6 years (0.89-14.04) and at start of targeted therapy was 11.62 years (3.64-15.42). All patients had previously a surgical procedure, and 12/13 had received another therapy prior BRAF inhibition: 11 chemotherapy (in one case, up to 4 different protocols) and 4 radiotherapy. Under targeted therapy, tumour response was obtained in 10 patients (size reduction equal to or greater than 25%), and best response was observed in the first 6 months of treatment in 7 children. Four patients progressed under treatment (all high-grade gliomas) and 2 progressed shortly after stopping the inhibitor (both low-grade gliomas). Five patients had grade 3-4 toxicity, with subsequent full recovery. A good and sustained clinical-radiological response, with acceptable tolerance, is described in patients with BRAFV600E mutated low-grade gliomas treated with BRAFV600E inhibitors. In contrast, the response in patients with high-grade gliomas was intermediate and of short duration, with early tumour progression.

La mutación puntual V600E del gen BRAF juega un papel fundamental en la tumorigénesis de muchos gliomas. La inhibición de su producto forma parte de terapias innovadoras emergentes en los últimos años. Conocer el rol de estos tratamientos resulta imprescindible. El objetivo del trabajo fue describir la respuesta clínico-radiológica en niños con gliomas BRAFV600E mutado tratados con inhibidores BRAF. Para ello se realizó un estudio descriptivo y retrospectivo en pacientes menores de 16 años con gliomas BRAFV600E mutado que recibieron vemurafenib o dabrafenib en el Hospital Garrahan. Trece pacientes tratados en los últimos 7 años fueron incluidos: 9 gliomas de bajo grado y 4 de alto grado. La mediana de edad al diagnóstico fue 8.6 años (0.89-14.04) y del comienzo del inhibidor 11.62 años (3.64-15.42). Inicialmente, todos habían realizado tratamiento quirúrgico, y 12/13 recibieron previamente otra terapia: 11 quimioterapia (eventualmente hasta 4 líneas distintas) y 4 radioterapia. Con la terapia dirigida, 10 pacientes tuvieron una disminución tumoral mayor o igual al 25%, quedando evidenciada en 7 niños la mejor respuesta dentro de los 6 meses del inicio. Hubo 4 progresados intratratamiento (todos alto grado), y 2 progresados prontamente luego de suspender el inhibidor (ambos bajo grado). Cinco presentaron efectos adversos grado 3-4, con recuperación ad-integrum. Se describe una buena y sostenida respuesta clínico-radiológica, con tolerancia aceptable, en pacientes con gliomas de bajo grado BRAFV600E mutado tratados con inhibidores BRAFV600E. En contraste, la respuesta en pacientes con gliomas de alto grado fue intermedia y de poca duración, con progresión tumoral precoz.

Terapias dirigidas con inhibidores del braf en gliomas pediátricos de alto y bajo grado con la mutación brafv600e. Experiencia del hospital Prof. Dr. Juan P. Garrahan / Targeted therapies with BRAF inhibitors for pediatric low- and high-grade gliomas with BRAF v600e mutation. Prof. Dr. Juan P. Garrahan Hospital experience

Resumen La mutación puntual V600E del gen BRAF juega un papel fundamental en la tumorigénesis de muchos gliomas. La inhibición de su producto forma parte de terapias innovadoras emergentes en los últimos años. Conocer el rol de estos tratamientos resulta imprescindible. El objetivo del trabajo fue describir la respuesta clínico-radiológica en niños con gliomas BRAF V600E mutado tratados con inhibidores BRAF. Para ello se realizó un estudio descriptivo y retrospectivo en pacientes menores de 16 años con gliomas BRAF V600E mu tado que recibieron vemurafenib o dabrafenib en el Hospital Garrahan. Trece pacientes tratados en los últimos 7 años fueron incluidos: 9 gliomas de bajo grado y 4 de alto grado. La mediana de edad al diagnóstico fue 8.6 años (0.89-14.04) y del comienzo del inhibidor 11.62 años (3.64-15.42). Inicialmente, todos habían realizado tratamiento quirúrgico, y 12/13 recibieron previamente otra terapia: 11 quimioterapia (eventualmente hasta 4 líneas distintas) y 4 radioterapia. Con la terapia dirigida, 10 pacientes tuvieron una disminución tumoral mayor o igual al 25%, quedando evidenciada en 7 niños la mejor respuesta dentro de los 6 meses del inicio. Hubo 4 progresados intratratamiento (todos alto grado), y 2 progresados prontamente luego de suspender el inhibidor (ambos bajo grado). Cinco presentaron efectos adversos grado 3-4, con recuperación ad-integrum. Se describe una buena y sostenida respuesta clínico-radiológica, con tolerancia aceptable, en pacientes con gliomas de bajo grado BRAF V600E mutado tratados con inhibidores BRAF V600E . En contraste, la respuesta en pacientes con gliomas de alto grado fue intermedia y de poca duración, con progresión tumoral precoz.

Abstract The BRAF V600E point mutation plays a key role in the tumorigenesis of many gliomas. Inhibiting its product is part of the innovative therapies emerging in recent years. Knowing the role of these treatments is essential. The aim of this experience was to describe the clinical-radiological response of pediatric BRAF V600E mutated gliomas treated with BRAF inhibitors. To this end, a descriptive and retrospective study was performed in patients under 16 years of age with BRAF V600E gliomas, who received vemurafenib or dabrafenib at Hospital Garrahan. Thirteen patients treated in the last 7 years were included: 9 were low-grade and 4 high-grade gliomas. The median age at diagnosis was 8.6 years (0.89-14.04) and at start of targeted therapy was 11.62 years (3.64-15.42). All patients had previously a surgical procedure, and 12/13 had received another therapy prior BRAF inhibition: 11 chemotherapy (in one case, up to 4 different protocols) and 4 radiotherapy. Under targeted therapy, tumour response was obtained in 10 patients (size reduction equal to or greater than 25%), and best response was observed in the first 6 months of treatment in 7 children. Four patients progressed under treatment (all high-grade gliomas) and 2 progressed shortly after stopping the inhibitor (both low-grade gliomas). Five patients had grade 3-4 toxicity, with subsequent full recovery. A good and sustained clinical-radiological response, with acceptable tolerance, is described in patients with BRAF V600E mutated low-grade gliomas treated with BRAF V600E inhibitors. In contrast, the response in patients with high-grade gliomas was intermediate and of short duration, with early tumour progression.

Targeting mitochondria in melanoma: Interplay between MAPK signaling pathway and mitochondrial dynamics.

Melanoma is a malignant proliferative disease originated in melanocytes, characterized by high metastatic activity and by the activation of oncogenes, such as B-RAF (40-60% of cases). Recent studies have shown that vemurafenib (a MAPK inhibitor) promoted disturbance of mitochondrial bioenergetics, although underlying mechanisms are not fully comprehended. Here we showed that MAPK inhibition by vemurafenib in B-RAFV600E-mutated human melanoma culminated in the inhibition of DRP1 phosphorylation, associated to a large mitochondrial network remodeling to the hyperfused phenotype, and increased oxidative phosphorylation capacity. Such alterations may be associated to melanoma resistance to vemurafenib, since the impairment of oxidative phosphorylation increased the vemurafenib cytotoxicity. These results point to the potential of mitochondrial dynamics as a targetable pathway in melanoma.

Vemurafenib downmodulates aggressiveness mediators of colorectal cancer (CRC): Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP), Protein Tyrosine Phosphatase 1B (PTP1B) and Transforming Growth Factor ß (TGFß).

Colorectal Cancer (CRC) therapy confronts challenges as chemoresistance and side effects. Therefore, drugs with antitumor properties that downmodulate aggressiveness mediators are required. Studies have shown the relevance of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP), Protein Tyrosine Phosphatase 1B (PTP1B), and Transforming Growth Factor ß (TGFß) in mediating proliferation, chemoresistance, and metastasis. In this study, we aimed to investigate the responsiveness of colorectal cancer lines (HT29 and HCT116) towards Vemurafenib and whether this treatment could modulate these aggressiveness mediators. Cytotoxicity Assays (MTT and Trypan Exclusion Test) were performed to evaluate the viability of HT29 and HCT116 cells treated with Vemurafenib. Western blotting was performed to analyze the amount and/or the activity of mediators (LMWPTP, PTP1B, TGFß, SMAD3), and the immunoprecipitation was performed to evaluate LMWPTP activity. This study brought up novel aspects of Vemurafenib action in colorectal cancer, which can decrease the activity of protein tyrosine phosphatases (LMWPTP and PTP1B) and the TGFß pathway, making them important in the CRC aggressiveness. By downmodulating colorectal cancer hallmarks, Vemurafenib appears as an interesting candidate for CRC therapeutic protocols.

Metalloproteinases Suppression Driven by the Curcumin Analog DM-1 Modulates Invasion in BRAF-Resistant Melanomas.

BACKGROUND: Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies. METHODS: Evaluated DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation. RESULTS: DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important Metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP-2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2. CONCLUSION: These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.

Metalloproteinases suppression driven by the curcumin analog DM-1 modulates invasion in BRAF-resistant melanomas

Background: Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies. Methods: Evaluated DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation. Results: DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important Metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP-2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2. Conclusion: These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.

ER stress promotes antitumor effects in BRAFi/MEKi resistant human melanoma induced by natural compound 4-nerolidylcathecol (4-NC).

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 µM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naïve, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.

Análise de custo por evento evitado (COPE) da associação de cobimetinibe e vemurafenibe e outras opções terapêuticas para o tratamento de melanoma metastático com mutação BRAF V600 / Cost of preventing an event (COPE) comparison between the association of cobimetinib and vemurafenib among other treatment options for metastatic melanoma with BRAF V600 Mutation

Objetivo: O melanoma maligno é o tipo de neoplasia de pele com pior prognóstico devido ao seu elevado potencial de gerar metástases e a sua letalidade. Algumas diretrizes internacionais recomendam a combinação de inibidores da via das proteínas quinases BRAF e MEK para o tratamento de melanoma metastático ou irressecável com mutação positiva BRAF V600 em primeira linha. Um estudo demonstrou que, em termos de sobrevida livre de progressão (SLP), a associação de cobimetinibe + vemurafenibe foi a opção terapêutica que proporcionou menor valor de número necessário para tratar (NNT) para o tratamento de melanoma metastático com mutação BRAF V600 em primeira linha. O objetivo desta análise foi de avaliar o custo por evento evitado (COPE) de cobimetinibe em associação com vemurafenibe no tratamento do melanoma irressecável ou metastático, positivos para mutações BRAF V600. Métodos: A análise econômica adotou a perspectiva do Sistema de Saúde Suplementar brasileiro. Os comparadores avaliados foram vemurafenibe monoterapia, dabrafenibe + trametinibe, nivolumabe e ipilimumabe. O COPE foi obtido pelo produto do NNT, baseado na SLP em 12 meses, reportado por Ho et al. e do custo de tratamento considerando custos de aquisição dos medicamentos e manejo de eventos adversos. Resultados: A maior eficiência da associação de cobimetinibe e vemurafenibe foi capaz de proporcionar o menor COPE (R$ 1.204.229) entre todos os comparadores da análise, demonstrando que, com um menor número de pacientes tratados, se observa um caso de sucesso com a terapia combinada, mesmo que esta apresente um custo anual de tratamento superior (R$ 626.477). Conclusão: A associação de cobimetinibe e vemurafenibe foi a mais eficiente em termos de NNT e COPE no melanoma metastático não tratado previamente.

Objective: Melanoma is a type of skin cancer with poor prognosis due to its high potential for metastasis and lethality. Some international guidelines recommend the combination of BRAF and MEK protein kinase pathway inhibitors for the first line treatment of metastatic or unresectable melanoma with BRAF V600 positive mutation. One study demonstrated that in terms of PFS, the combination of cobimetinib + vemurafenib was the first line therapeutic option that provided the lowest number needed to treat (NNT) for BRAF V600 mutated metastatic melanoma. The objective of this analysis was to evaluate the cost of preventing and event (COPE) of cobimetinib in combination with vemurafenib in the treatment of unresectable or metastatic melanoma, positive for BRAF V600 mutations. Methods: The economic analysis adopted the perspective of the Brazilian Supplementary Health System. The comparators evaluated were vemurafenib monotherapy, dabrafenib + trametinib, nivolumabe and ipilimumab. COPE was obtained from the NNT product, based on 12-month progression-free survival (PFS), reported by Ho et al., and the cost of treatment considering medication acquisition costs and adverse event management. Results: The greater efficiency of the combination of cobimetinib and vemurafenib was able to provide the lowest COPE (R$ 1,204,229) among all comparators of the analysis, demonstrating that with a lower number of treated patients a case of success was observed with the combination therapy, even if it presents a higher annual cost of treatment (R$ 626,477). Conclusion: The combination of cobimetinib and vemurafenib was the most efficient in terms of NNT and COPE for previously untreated metastatic melanoma.

Galectin-3 sensitized melanoma cell lines to vemurafenib (PLX4032) induced cell death through prevention of autophagy.

Melanoma is a current worldwide problem, as its incidence is increasing. In the last years, several studies have shown that melanoma cells display high levels of autophagy, a self-degradative process that can promote survival leading to drug resistance. Consequently, autophagy regulation represents a challenge for cancer therapy. Herein, we showed that galectin-3 (Gal-3), a ß-galactoside binding lectin which is often lost along melanoma progression, is a negative regulator of autophagy in melanoma cells. Our data demonstrated that Gal-3low/negative cells were more resistant to the inhibition of the activity of the cancer driver gene BRAFV600E by vemurafenib (PLX4032). Interestingly, in these cells, starvation caused further LC3-II accumulation in cells exposed to chloroquine, which inhibits the degradative step in autophagy. In addition, Gal-3 low/negative tumor cells accumulated more LC3-II than Gal-3 high tumor cells in vivo. Resistance of Gal-3low/negative cells was associated with increased production of superoxide and activation of the Endoplasmic Reticulum (ER) stress response, as evaluated by accumulation of GRP78. Pharmacological inhibition of autophagy with bafilomycin A reversed the relative resistance of Gal-3low/negative cells to vemurafenib treatment. Taken together, these results show that the autophagic flux is dependent on Gal-3 levels, which attenuate the prosurvival role of autophagy.

Rescate quirúrgico de metástasis suprarrenal por melanoma, previo tratamiento con vemurafenib / Surgical rescue of adrenal metastasis by melanoma following treatment with vemurafenib

Presentamos el caso de una paciente con metástasis suprarrenal única de melanoma, que fue some-tida a tratamiento sistémico con vemurafenib (un inhibidor de la quinasa BRAF) en primera instancia y luego a tratamiento quirúrgico. La bibliografia actual recomienda el tratamiento quirúrgico de las metástasis de melanoma y sostenen que, en aquellos pacientes con mutación de la quinasa BRAF, los inhibidores de esta serían seguros acompañando a las metastasectomías y mejorarían el control de la enfermedad de forma más sostenida en el tempo. Conclusión: si bien, durante muchos años, la cirugía fue la única terapia para la enfermedad en estadio IV, con las terapias de drogas modernas la situación se ha vuelto más compleja; esto ha creado el desafo de determinar la combinación óptma y la secuencia de terapias adecuada; en este sentido consideramos fundamental que casos como el presentado aquí se discutan en comité de tumores.

We report the case of a patent presenting with a single melanoma metastasis in the adrenal gland, who underwent systemic treatment with vemurafenib (BRAF kinase inhibitor) in the frst instance and then underwent surgery. Current literature recommends surgical treatment of melanoma metastases and supports the use of inhibitors in patents with a BRAF kinase mutaton since they may improve disease control in a more sustained manner over tme. Although for many years surgery was the only therapy for stage IV disease, with modern drug therapies, the situaton has become more complex; this has created the challenge of determining the optmal combinaton and appropriate sequence of therapies; in this sense we consider it fundamental that cases such as the one presented here are discussed in tumor commitee meetings.

Infección necrotzante de la pared abdominal posticesárea / Necrotzing infecton of the abdominal wall following a cesarean secton

La infección necrotzante de partes blandas (INPB) es una entidad rara, que afecta principalmente a individuos con factores de riesgo específicos pero en ocasiones suele afectar a mujeres puérperas, que al poseer un estado de inmunosupresión favorecería la aparición y curso más agresivo de la enfermedad. Presentamos el caso de una mujer de 20 años edad puérpera, sin antecedentes patológicos, que desarrolla una fascits necrotzante de la pared abdominal como complicación de una cesárea. Se realizó una resección amplia de la pared abdominal, anexohisterectomía, lavado de cavidad peritoneal y cierre temporal con bolsa de Bogotá. Tras una internación prolongada en unidad de terapia intensiva se programó el cierre de la pared abdominal utlizando una malla de poliglactina en contacto con las vísceras, sobre la cual se colocó una malla de polipropileno para proporcionar resistencia. Se inicia terapia de presión negativa (TPN) con vacuum pack de Barker hasta programar la reconstrucción de la pared abdominal con injertos de piel autólogos. Luego se continúo tratamiento con sistema V.A.C. ® ATS sobre los injertos, presentando una evolución favorable. El objetivo de esta carta científica es presentar una complicación poco habitual de la operación cesárea, destacando los aspectos más importantes de la enfermedad, definiendo algunas recomendaciones y remarcando la importancia del abordaje multidisciplinario.

Necrotzing sof tssue infecton (NSTI) is a rare disease that mainly afects individuals with specific risk factors but sometmes afects postpartum women as well. These women have a state of immunosup-pression, which would favor the appearance and more aggressive course of the disease. We report the case of a 20-year-old woman with no pathological history who developed necrotzing fasciits of the abdominal wall as a complicaton of a cesarean secton. An extensive abdominal wall resecton was performed, followed by total histerectomy and bilateral adnexectomy, peritoneal cavity lavage, and temporary abdominal closure with a Bogotá bag. Afer prolonged hospitalizaton in an intensive care unit, the abdominal wall was closed using a polyglactin mesh in contact with the viscera, on which a polypropylene mesh was placed to provide resistance. Negative pressure wound therapy with a Barker´s vacuum pack was started untl the reconstructon of the abdominal wall with autolo-gous skin grafis could be programmed. Then, a V.A.C. ATS ® therapy was used on skin grafis, showing a favorable evoluton. The objective of this scientific leter is to discuss an uncommon complicaton of the cesarean secton, emphasizing the most important aspects of the disease, and defining recommendatons for the mana-gement of this pathology and its multidisciplinary approach.

Characteristics of adverse drug reactions in a vemurafenib early post-marketing phase vigilance study in Japan.

BACKGROUND: Post-approval research or monitoring is important to determine real-world safety of new products; however, evidence is scant for vemurafenib in Japanese patients. In Japan, a unique system is officially obligated to investigate post-approval safety. Here we report the first adverse drug reaction (ADR) data from vemurafenib-treated Japanese patients with metastatic melanoma. Data were collected in an early post-marketing phase vigilance (EPPV) study. METHODS: ADRs were events for which a causal relationship with vemurafenib could not be ruled out or was unknown. ADR data were collected for patients treated with vemurafenib (960 mg bid) between 26 February and 25 August 2015. RESULTS: Among 95 patients, 46 patients had 118 ADRs (24 serious ADRs in 13 patients). The most common serious ADRs were hypersensitivity (n = 1; 3 events), arthralgia (n = 2; 2 events), pyrexia (n = 2; 2 events) and drug eruption (n = 2; 2 events). Seven patients had serious skin disorders or hypersensitivity, six of whom had prior anti-programmed cell death-1 (PD-1) antibodies 5-35 days before starting vemurafenib. ADR reports of serious skin disorders appeared to be collected more rapidly than previously reported. Cutaneous squamous cell carcinoma developed in only one patient. CONCLUSIONS: EPPV in Japanese vemurafenib-treated patients identified no new safety signals. The most serious skin and hypersensitivity ADRs occurred in patients with prior anti-PD-1 exposure. Cutaneous squamous cell carcinoma appeared to be rare in Japanese patients. Further research is needed to clarify whether prior treatment with anti-PD-1 agents or racial differences affect the characteristic profile of cutaneous ADRs in Japanese patients.